The invention relates to a method and a kit for diagnosing a molecular phenotype of a patient suffering from an illness accompanied by chronic inflammation as well as a medicament for treating such a patient.
Chronic inflammations constitute an increasing medical problem area of high socioeconomic significance. This includes in particular the following groups of illnesses: autoimmune diseases and diseases from the area of rheumatic diseases (manifestations among others on the skin, lungs, kidneys, vascular system, nervous system, connective tissue, locomotor system, endocrine system), immediate-type allergic reactions and asthma, chronic obstructive lung diseases (COPD), arteriosclerosis, psoriasis and contact eczema and chronic rejection reactions after organ and bone marrow transplants. Many of these diseases are showing a rising prevalence in the last decades not only in industrial nations, but sometimes around the world. For example, in Europe, North America, Japan and Australia more than 20% of the population suffers from allergic diseases and asthma. Chronic obstructive lung diseases are currently the fifth most frequent cause of death throughout the world and according to calculations of the WHO they will represent the third most frequent cause of death in the year 2020. Arteriosclerosis with the secondary diseases of cardiac infarction, stroke and peripheral arterial disease leads the world in morbidity and mortality statistics. Together with neurodermatitis, psoriasis and contact eczema are in general the most frequent chronic inflammatory diseases of the skin.
Due to the interactions between environmental factors and a genetic disposition, which are to date only poorly understood, there are sustained dysregulations of the immune system. In this connection the following common principles can be established for these different diseases:
(A) An excessive immune response to what are ordinarily harmless antigens for human beings. These antigens can be components of the environment (e.g. allergens such as pollen, animal hairs, food, mites, chemical substances such as preservatives, dyestuffs, detergents). In these cases patients develop an allergic reaction. In the case of e.g. active and passive smokers, chronic pulmonary lung diseases (COPD) develop. On the other hand, the immune system can also react against components of its own organism, recognize them as foreign and initiate an inappropriate inflammatory response. In these cases an autoimmune illness develops. In any case, harmless, non-toxic antigens are erroneously as foreign or dangerous and an inappropriate inflammatory response is set in motion.
(B) The diseases run in phases, including initiation, progression of the inflammatory response and the associated destruction and reconstruction with loss of organ functionality (so-called remodeling).
(C) The diseases show patient-specific sub-phenotypic manifestations.
(D) Components of the innate and acquired immunity have a sustained involvement in the initiation, maintenance and destructive and remodeling processes. Under the influence of the innate immunity (important components: antigen-presenting cells with their diverse populations and the complement system) there is an activation and differentiation of the cells of the adaptive immune system (important components: T and B lymphocytes)> The T cells take over central functions in the further course by differentiating in highly specialized effectors.
In this connection they activate and acquire certain effector mechanisms, including, in particular the following functions: antibody production: control of the functionality of effector cells of the immune system (e.g. such as neutrophilic, basophilic, eosinophilic granulocytes), feedback to functions of the innate immune system, influencing of the functionality of non-hematopoietic cells such as e.g. epithelial, endothelial, connective tissue, bones and cartilage and above all neuronal cells. Here there is a special interaction between immune and nervous systems, from which the concept of neuro-immunological interaction in the case of chronic inflammations developed.
Since the T cells, which have already been mentioned, take over central functions in the course of the disease, an understanding of their specialization is critical. A complex signal transduction cascade is involved in the differentiation of naïve CD4+ cells to Th1 or Th2 cells.
The stimulation via the T cell receptor through the corresponding peptide MHC complex induces clonal expansion and programmed differentiation of CD4+ T lymphocytes to T helpers (Th) 1 or Th2 cells. The differentiation of these two sub-types occurs on the basis of their cytokine profiles. Th1 cells produce interferon-γ (INFγ), interleukin 2 (IL-2) and tumor-necrosis-factor-α, while Th2 cells secrete IL-4, IL-5, IL-9 and IL-13. Bacterial and viral infections induce an immune response which is dominated by Th1 cells. On the other hand Th2 cells regulate igE production against parasites. In the process there is a balance between Th1 and Th2 cells. The destruction of this balance causes diseases, so an excessive Th1 cell response is associated with autoimmunity diseases, while allergic diseases are at the basis of a reinforced Th2 cell response.
It is known that Th1 cytokines are involved in the pathogenesis of autoimmune diseases such as e.g. autoimmune uveitis, experimental allergic encephalomyelitis, type 1 diabetes mellitus or Crohn's disease, while Th12 cytokines (IL-4, IL-5, IL-13 or IL-9) are involved in the development of chronic inflammatory respiratory ailments, such as e.g. airway eosinophilia, asthma, mucus hypersecretion and airway hyperresponsiveness. These diseases are brought about by pathophysiological changes during the production of characteristic cytokines by antigen-specific Th cells. Th2 cell sub-populations in the lungs and the airways cause the characteristic symptoms of bronchial asthma in the animal model
Among other things, two transcription factors are involved in the development of autoimmune diseases and chronic inflammatory reactions: the Th1 cell-specific transcription factor Tbet and the Th2 cell-specific transcription factor GATA-3.
The Th1 cell-specific transcription factor Tbet is primarily responsible for the differentiation of naïve CD4+ T cells to Th1 cells. Its expression is controlled via the signal transduction pathways of the T cell receptor (TZR) and via INFγ receptor/STAT1. Tbet transactivates the endogenous INFγ gene and induces INFγ production. The in vivo function of Tbet is confirmed in knock-out mice (Tbet−/−). The quantity of Th2 cytokines is increased in mice that are deficient in Tbet.
The function of Tbet in mucosal T cells is known in the development of inflammatory bowel diseases. The transcription factor Tbet specifically induces the development of Th1 cells and controls the INFγ production in these cells. Through the inhibition of Tbet the balance between Th1 and Th2 cells is shifted in favor of the Th2 cells.
Many inflammatory diseases on the other hand, such as allergic asthma for example, are associated with an activation of Th2 cells. Th2 cells have an essential function in the development of allergic diseases, in particular various asthma ailments. The differentiation of Th0 cells to Th2 cells necessary for this is dependent on the transcription factor GATA-3. GATA-3 is a member of the GATA family of transcription factors.
The Th2 cell-specific transcription factor GATA-3 is primarily responsible for the differentiation of naïve CD4+ T cells to Th2 cells. In the process, the Th2 cell differentiation is primarily controlled by two signal transmission pathways, the T cell receptor (TZR) and the IL-4 receptor pathway: Signals forwarded from TZR activate the Th2 cell-specific transcription factors cMaf and GATA-3 as well as also the transcription factors NFAT and AP-1. The activation of the IL-4 receptor results in the binding of STAT6 on the cytoplasmic domain of the IL-4 receptor, where it is phosphorylated by Jak1 and Jak3 kinases. The phosphorylation for its part results in the dimerization and translocation of STAT6 to the nucleus, where STAT6 activates the transcription of GATA-3 and other genes. GATA-3 is a zinc finger transcription factor which is expressed exclusively in mature Th2 cells, not in Th1 cells.
Th2 cells produce cytokines such as for example IL-4, IL-5, IL-6, IL-13 and GM-CSF. The polarization to Th2 inhibits a Th1 differentiation through suppression of Tbet and vice versa. However, the expression of GATA-3 is not restricted to T cells. An expression of GATA-3 was also able to be confirmed in eosinophilic and basophilic granulocytes, mast cells and epithelial cells. GATA-3 plays a central role in the immunopathogenesis of chronic inflammatory diseases, in particular of allergic asthma.
Established preparations for the treatment of chronic inflammatory diseases are among others Corticosteroids, anti-leukotrienes, immunosuppressives and Anti-IgE monoclonal antibodies. Asthma patients however respond with varying degrees of success to these therapeutic agents. For a long time the question of what these differences in effectiveness were to be attributed to has remained unresolved. As a consequence, the appropriate therapy had to be fine-tuned on the patient more or less in accordance with the principle of “trial and error”.
However, only recently was it determined that patients suffering from asthma, for example, could be further divided into subgroups (Woodruff et al., 2009, T-helper Type 2-driven inflammation Defines Major Subphenotypes of Asthma, Am J RespiCrit Care Med, Vol 180, 388-395). Thus, it was shown that there are at least two sub-groups of asthma patients, which were designated as “Th2 high” and “Th2 low”. The subgroup “Th2 high” in the process has an increased expression of the POSTN gene, which codes for the protein periostin, as well as the genes for IL-3 and IL-5. The group “Th2 low” of tested asthma patients shows a low POSTN gene expression, comparable to a control group of healthy persons. These differing molecular phenotypes could be one cause for the different effectiveness of common therapeutic agents. Thus for the subgroup “Th2 high” an improved treatment response to treatment with corticosteroids was determined.
It was also found that two groups of asthma patients, namely “Th2 high” and “Th2 low”, respond with varying degrees of success differently to a therapy with a humanized monoclonal antibody to IL-13 (Corren et al., 2011, Lebrikizumab Treatment in Adults with Asthma, The New England Journal of Medicine, 10.1056/NEJMoa 1106469). In the process, an empirical classification of asthma patients in the group “Th2 high” occurred first, when the values for serum-IgE were higher than 100 IU/ml and the number of eosinophilic granulocytes was at 0.14×109 cells per liter or greater. With corresponding values below these patients were placed in the group “Th2 low”. Alternatively, there was a classification by the serum periostin level, which serves as a surrogate marker for Th2 cytokine IL-13, which is difficult to establish in blood or airway samples. In the process, the fact that IL-13 among others induces in vitro the expression of the periostin coding gene POSTN in epithelial cells (Woodruff et al., 2007, Proc Natl Acad Sci USA, 104(40):15858-63. Genome-wide profiling identifies epithelial cell genes associated with asthma and with treatment response to corticosteroids). In accordance with Corren et al., 2011, patients with a serum periostin level above the average were placed in a “periostin high” group. For the mentioned sub-groups “Th2 high” and “Periostin high” a better treatment response to treatment with Anti-IL-13 antibodies by tendency was described.
According to WO 2009/124090 A1, a certain classification of asthma patients is likewise proposed, wherein the gene expression of a plurality of candidate genes, such as for example POSTN, CLCA1 and SERPINB2 is employed. Since it is known that this gene is highly regulated by the Th2 cytokine IL4 or IL-13, the cluster is also referred to as “IL-4/IL-13 signature”. Along with the measurement of the serum periostin level as well as the corresponding mRNA quantity, in the process a determination of the values for serum IgE and the number of eosinophilic granulocytes were also described.
One disadvantage of patient stratification on the basis of this gene expression, above all of POSTN, is the fact that along with an “IL-4/IL-13 signature”, the cytokine IL-5 also plays a significant role in the genesis of asthma. In addition, the role of the protein periostin in the immune cascade and thus the pathogenesis is unknown.
Thus the problem arises of finding a biomarker that is suitable for reliable and simultaneously clinically practicable molecular phenotyping of a human patient who is suffering from a disease that is accompanied by chronic inflammations in the groups “Th2 high” or “Th2 low” or “Th1 high” or “Th1 low”. In addition, the patient classified in this manner should be able to be treated with a therapeutic agent that is especially effective specifically for this subgroup. The biomarker should make possible/facilitate an individual prediction about the effectiveness of a therapeutic agent with respect to a patient, in particular an asthma patient.